The Role And Mechanism Of Roscovitine In Mice With Oxygen Induced Retinal Neovascularization

Part1 The expression of p35 in oxygen induced retinopathy and the effect of roscovitine on retinal neovascularizationObjective: Establish oxygen-induced retinopathy(OIR)mouse models.Investigate the expression of cyclin-dependent kinase 5 activator subunit 1(p35)in the mouse model of OIR.And investigate the effect of roscovitine,an inhibitor of cyclin dependent kinase 5(Cdk5),on retinal neovascularization.Method: Forty C57BL/6J newborn mice,male and female unlimited,were randomly divided into 6 groups.1.Blank control group,were fed in normal environment without special treatment.2.OIR group,were dealt with standard model of OIR.3.OIR solvent group,were dealt with standard model of OIR.Both eyes was given 0.5μl solvent(for dissolving roscovitine)by intravitreal injection of 12 days old.4.OIR drug intervention group comprise three doses.They were both dealt with standard model of OIR,and was given 0.5μL×0.01μg/μL,0.5μL×0.10μg/μL,0.5μL×1.00μg/μL roscovitine solution by intravitreal injection of 12 days old.OIR models are established by reference to Smith’s standard method.At 17 days old,the mice were executed and the eyeballs were removed.The number of endothelial cells breaking through the inner limiting membrane in each group was observed by hematoxylin-eosin(HE)staining.The whole retina was stained with griffonia simplicifolia isoform B4(GS-I-B4)to observe the morphology of retinal blood vessels.The avascular areas and neovascularized areas were calculated.And we compared with the relative concentration of p35 in retina of blank control group and OIR group by Western blot.Results: The results of Western blot showed that the relative concentration of p35 protein in the retina of OIR group was significantly higher than that the blank control group.There were no endothelial cells breaking through the internal limiting membrane in the blank control group,and a large number of endothelial cells breaking through the internal limiting membrane in the OIR group and the OIR solvent group.In the three drug doses of the oxygen induced drug intervention group,the numbers of endothelial cell nuclei breaking through the inner boundary membrane decreased,and were correlated with roscovitine concentration.GS-I-B4 staining showed that the retinal vascular network in the blank control group developed normally.In the OIR group and the OIR solvent group,there were obvious pathological changes in the retina.Avascular areas were formed in the central area of the retina,branch vessels were tortuous,and neovascularization grow.In OIR drug intervention group,the avascular areas and neovascularized areas of three doses were significantly reduced,and there was a significant correlation with drug concentration.Conclusion: P35 expression increases in mice with oxygen induced retinal.Roscovitine,an inhibitor of Cdk5 inhibitor,inhibited retinal neovascularization in OIR mice,promoted retinal vascular normalization,and the inhibitory effect was positively correlated with roscovitine concentration in a certain range.Part2 The mechanism of roscovitine inhibiting retinal neovascularization and its effect on the mouse retinal structureObjective: To investigate the effect of roscovitine on the protein concentration of phospho-Akt s473(p-Akt s473),hypoxia inducible factor-1α(HIF-1α)and vascular endothelial growth factor a(VEGF-A)in the retina of mice induced by oxygen,and to explore the mechanism of roscovitine inhibiting retinal neovascularization.The effect of roscovitine on the retinal structure of mice was also investigated.Method: Twenty-five C57BL/6J newborn mice,male and female unlimited,were randomly divided into 5 groups.1.Blank control group,were fed in normal environment without special treatment.2.OIR group,were dealt with standard model of OIR.3.OIR solvent group,were dealt with standard model of OIR.Both eyes was given 0.5μl solvent(for dissolving roscovitine)by intravitreal injection of 12 days old.4.OIR drug intervention group,were dealt with standard model of OIR.Both eyes were given 0.5μL×1.00μg/μL roscovitine solution by intravitreal injection of 12 days old.5.Normoxia drug intervention group,were fed in normal environent,and were given 0.5μL×1.00μg/μL roscovitine solution by intravitreal injection of 12 days old on binoculus.OIR models are established by reference to Smith’s standard method.At 17 days old,the mice were executed and the eyeballs were removed.We compared with the relative concentration of HIF-1α、VEGF-A、AKT、p-AKT s473 in retina of each group by Western blot.HE staining was used to measure and compare the thickness of the whole retinal layer and the outer nuclear layer of the blank control group and the normoxia drug intervention group.Results: There was no significant difference in the relative concentrations of p-Akt s473,HIF-1α and VEGF-A protein between the OIR group and OIR solvent groups.Thetwo groups were significantly higher than the blank control group.After roscovitine intervention,p-Akt s473,HIF-1α and VEGF-A protein in the oxygen-induced drug intervention group were significantly lower than that in the oxygen-induced solvent control group.The thickness of the whole layer and the outer nuclear layer of the retina have no significant difference between the blank control group and the normoxia drug intervention group.The structure of the retina was normal.Conclusion: Roscovitine may play an important role in improving the retinal vessels of OIR mice by inhibiting the phosphorylation of Akt in ser473,and affecting the expression of HIF-1 α and VEGF-A.There was no effect on the structure of retina in mice by injecting with 0.5μL×1.00μg/μL roscovitine.